This proposal seeks support for a long-standing translational grant funded continuously by NICHD for the past 22 years, serving as the primary vehicle for work investigating the pathogenesis of IR in adolescent obesity. We described a distinct endophenotype in obese adolescents characterized by a thin superficial layer of abdominal subcutaneous adipose tissue (SAT), increased visceral adipose tissue (VAT), marked IR, dyslipidemia, and fatty liver. During the last cycle of the grant, we began to unravel the cellular/molecular mechanisms associated with this phenotype and its relations to IR. We demonstrated a key role of impaired subcutaneous abdominal adipogenesis in the pathogenesis of IR in obese adolescents supporting the hypothesis that the ability to retain fat in the subcutaneous depots seems to be associated with decreased VAT and reduced ectopic fat deposition and a more favorable metabolic profile. Building on these findings we propose 3 major hypotheses: Hypothesis 1: The reduced transcription of key lipogenic/adipogenic genes in abd SAT translates into reduced in vivo TG synthesis and adipocyte proliferation, which in turn will contribute to ectopic fat accumulation and IR in obese adolescents with a High VAT/SAT ratio. Hypothesis 2: The storage capacity of gluteal SAT is a determinant for the level of VAT/SAT fat distribution in obese adolescents. Approach for Hypothesis 1 & 2: We will employ the novel method of 2H2O to quantify in vivo TG synthesis, and adipocyte proliferation. This method will be added to measures of adipocyte size distribution, gene expression, regional fat distribution (MRI & DEXA) and clamp measures of insulin sensitivity in obese adolescents with a high VAT/SAT ratio compared to an age, gender and pubertal stage matched group with a low VAT/SAT ratio. Hypothesis 3A: High dietary intake of palmitate is associated with an increased ceramide content which in turn might upregulate the inflammasome complex in the SAT thereby contributing to IR in obese adolescents. Hypothesis 3B: To explore if reducing the dietary intake of palmitate will decrease the production of ceramide in the SAT which will mitigate the activation of the inflammasome complex and reduce IR in obese adolescents with a High VAT/SAT ratio. Approach for Hypothesis 3A&3B: The plasma and SAT (abd & gluteal) sphingolipid concentrations will be determined by LC-MS/MS, by Dr. Scherer at UTSW and related to the intake of palmitate intake obtained by a three day dietary intake. Activation of caspase-1 protein will be measured by western blot (Dr. Dixit's lab at Yale) and transcriptomic of regulatory genes of caspase-1 by qPCR, which will be measured before and after two months of a randomized parallel control study with a low palmitate/saturated fat dietary intervention. The studies outlined here will provide a better understanding of IR in adolescent obesity. The grant will accomplish these aims through unifying a collaborative interdisciplinary team of world-renowned scientists at Yale spanning the breadth of disciplines from clinical sciences (Pediatrics) to basic sciences (Immunology, Adipose biology) to Nutritional Sciences (UC Berkeley and UVM).